Standardization of SARS-CoV-2 Nucleic Acid Amplification Techniques by Calibration and Quantification to the First WHO International Standard for SARS-CoV-2 RNA

Clinical decision-making regarding isolation of SARS-CoV-2 patients is usually based on semiquantitative cycle-threshold (Ct) values without standardization. However, not all molecular assays produce Ct values, and there is ongoing discussion about whether Ct values can be safely used for decision-making. In this study, we standardized two molecular assays which use different nucleic acid amplification techniques (NAAT): the Hologic Aptima SARS-CoV-2/Flu (TMA) and Roche Cobas 6800 SARS-CoV-2 assays. We calibrated these assays against the first WHO international standard for SARS-CoV-2 RNA by using linear regression of log10 dilution series. These calibration curves were used to calculate viral loads for clinical samples. Clinical performance was assessed retrospectively using samples collected between January 2020 and November 2021, including known positives of the wild-type SARS-CoV-2 virus, the VOCs (alpha, beta, gamma, delta, and omicron) and quality control panels. Linear regression and Bland–Altman analysis showed good correlations for SARS-CoV-2 between Panther TMA and Cobas 6800 when standardized viral loads were used. These standardized quantitative results can benefit clinical decision-making and standardization of infection control guidelines.


Introduction
Since the beginning of the SARS-CoV-2 pandemic in 2019, over 575 million confrmed cases and over 6.3 million deaths have been reported worldwide (WHO Coronavirus (COVID-19) Dashboard, 2 August 2022). Te burden of disease resulted in reorganization of hospitals in order to care for COVID-19 patients, and in many hospitals, the treatment of other diseases was downscaled and noncritical care was limited to urgent cases only [1]. From the beginning of the pandemic, the gold standard for detection of SARS-CoV-2 infection has been nucleic acid amplifcation techniques (NAAT) such as RT-PCR. A complicating factor has been the observation that individuals can remain PCR-positive for a considerable amount of time, even when the patient has recovered and symptoms have disappeared [2]. Studies early in the pandemic showed that prolonged RT-PCR positivity did not correlate with presence of the infectious virus [3,4], which led to the practice of using semiquantitative RT-PCR results, such as cycle-threshold (Ct) values to guide infection prevention measures and release of patients from isolation [5]. Since Ct values can difer signifcantly between diferent tests and laboratories, there is ongoing discussion whether Ct values can be reliably used to set clinical cutofs [6]. In addition, other NAATs such as transcription mediated amplifcation (TMA) and loopmediated isothermal amplifcation (LAMP) do not provide semiquantitative values. Te Hologic Aptima SARS-CoV-2/ Flu assay combines the technologies of target capture and real-timetranscription-mediated amplifcation (TMA) and produces "time to positivity" (TTP) measurements that in principle could be used as semiquantitative results. We have standardized the Hologic Aptima SARS-CoV-2/Flu and Roche Cobas 6800 SARS-CoV-2 assays for detection of SARS-CoV-2 by calibrating these assays to the frst international standard for SARS-CoV-2 RNA and have evaluated the quantitative results.
Clinical performance was evaluated retrospectively with respiratory tract samples collected and stored at −80°C between January 2020 and November 2021. Tis included known positives of wildtype SARS-CoV-2 and the variants of concern (VOC) alpha, beta, gamma, delta, and Omicron. Due to logistic constraints, there was one additional freeze/ thaw cycle before testing on the Panther TMA and Gen-eXpert systems, compared to Cobas 6800.
Pretreatment of sputa (SP) was performed as described before [9,10]. Troat and nasal swabs were not pretreated. Samples (500 μL) were added directly to the Panther Fusion Specimen Lysis tubes, containing 710 μL specimen transport media (STM). According to the manufacturer, Panther TMA results are qualitative, but also time-to-positivity (TTP) results are reported as a semiquantitative measure. Tis is the time, given in minutes with one decimal after the decimal point, necessary for the fuorescent signal to reach a specifed detection threshold. In this study, we have used these TTP results to correlate the measurements to the quantitative international WHO RNA standard. For the Cobas 6800 SARS-CoV-2 assay, samples were inactivated prior to the analysis by adding 500 μL of the sample to 750 μL Magna-Pure 96 external lysis bufer (Roche) as per the routine protocol for SARS-CoV-2 detection in our laboratory. Te Roche Cobas 6800 SARS-CoV-2 assay targets the ORF1a/ b nonstructural region (target 1) and the pan-SarbecoE-gene region (target 2). Calibration to the quantitative international WHO RNA standard was performed with the Ct values of the ORF1a/b nonstructural region (target 1). Discrepant samples were retested in the Xpert Xpress SARS-CoV-2/Flu/RSV plus assay (GeneXpert, Cepheid) [11,12].

. Results
Analytical sensitivity of Panther TMA was 9.17E1 IU/mL (range 3.59E1-9.66E3), determined by probit analysis at the 95% hit rate, while analytical sensitivity of the Cobas 6800 ORF1a/b assay was 1.82E2 IU/mL (range 1.07E2-3.56E3), resulting in a diference of 0.30 log10. In the SARS-CoV-2 RIVM EQA panel, the Cobas 6800 ORF1a/b assay scored 100%. One sample was not detected by the Panther TMA assay, compared to the expected results, whereas the Cobas 6800 pan-Sarbeco assay failed to detect two samples. One additional sample was detected in the Cobas 6800 pan-Sarbeco assay which, according to the panel composition information, contained SARS-CoV-1.
In Figure 1(a), Ct values from the Cobas 6800 ORF1a/b nonstructural region were plotted against the TTP values from the Panther TMA assay. Only samples positive for both the Cobas 6800 ORF1a/b and Panther TMA assays were included in the regression analysis. As expected, results showed a deviation from zero in both Deming regression and the Bland-Altman plot, confrming that these results cannot be compared without standardization. For standardization calibration, curves were constructed by linear regression for the Panther TMA (y � −2.0833, x + 24.897, and R 2 � 0.9391) and Cobas 6800 ORF1a/b assays (y � −2.8507, x + 43.461, and R 2 � 0.9782) using the results of the log10 dilution series from the internal standard. Tese calibration curves were used to calculate the viral loads of the samples from the clinical evaluation and the RIVM EQA panel in IU/ml. Deming regression analysis, when IU/ml values were used (Figure 1(b)), showed a good correlation (slope 0.9725, R 2 � 0.8903) between the TMA and Cobas

Discussion
Tere are limited studies that are routine SARS-CoV-2 molecular assays which are calibrated and standardized to international standards. Sahoo et al. described comparison of GeneXpert to a laboratory-developed test using the frst WHO international RNA standard and showed harmonization and standardized quantifcation [13]. Using a similar approach, we were able to calibrate the semiquantitative measurements from the Panther Aptima SARS-CoV-2 real-time TMA and Cobas 6800 SARS-CoV-2 ORF1a/b assays to a relevant quantitative standard. Dilution series showed linear correlations with an analytical sensitivity of 9.17E1 IU/ml for Panther TMA and 1.82E2 for Cobas 6800 ORF1a/b assays, respectively. Overall, clinical sensitivity of Panther TMA was 100%, whereas the clinical specifcity was 93.1% compared to that of the Cobas 6800 ORF1a/b and pan-Sarbeco assays. Since only two samples were positive for Panther TMA and negative for the Cobas 6800 ORF1a/ b and pan-Sarbeco assays, this number should be taken with caution. Both samples were positive using the additional geneXpert assay; however, with high Ct values. Tis suggests a slightly higher clinical sensitivity for Panther TMA compared tothan for the Cobas 6800 ORF1a/b and pan-Sarbeco assays. However, additional analysis of samples with a low amount of the virus close to the detection limit of both assays is needed to draw more frm conclusions.
Deming regression and Bland-Altman analysis showed comparable results between the Panther TMA and Cobas 6800 ORF1a/b assays when calibrated IU/ml values were used. Noncalibrated values gave a deviation from zero, showing that the TTP and Ct values are not comparable and that calibration is necessary to gain reliable quantitative and comparable results.
Detection of SARS-CoV-2 can be performed on various sample types. Te Panther TMA assay is intended for use with nasal and/or throat swabs only, the preferred sample type for SARS-CoV-2 detection at our hospital. However, for SARS-CoV-2 patients in the intensive care ward, sputa are also used. Pretreated samples of this sample type were included in the clinical study without problems.
Since the introduction of international standards in molecular diagnostics, laboratories have been able to produce reliable and comparable quantitative results between assays and laboratories, which opened the opportunity to international interlaboratory studies [14,15]. Currently, quantitative molecular assays are fully integrated in the monitoring of infectious diseases. However, most quantitative assays have only been validated on homogeneous samples such as serum and plasma. Quantifcation of heterogeneous samples like respiratory tract samples is more challenging since also variations in sample composition, sample collection, and transport media can infuence quantitative results. Although we report here that quantifcation to RNA standards is possible, we also stress caution with interpretation of these quantitative results due to the intrinsic variable nature of respiratory samples.
At the Erasmus Medical Centre, a tertiary care hospital, patients infected with SARS-CoV-2 are treated in oneperson isolation rooms by healthcare workers using personal protective equipment. It is known that infection control measures can lead to decreased attention given to those who are treated in isolation and can have negative psychological efects on the patient. In our hospital, infection control measures for SARS-CoV-2 are lifted for patients admitted at the general ward after a minimum disease duration of 10 days, at least 2-3 days improvement of clinical symptoms and negative PCR or PCR with a high Ct value (Ct value > 32, [3]). Based on our studies, this Ct value is comparable with a viral load of <1.0E4 IU/ml. Following introduction of the calibrated quantitative realtime Panther TMA assays for SARS-CoV-2 in our laboratory, this viral load cutof of 1.0E4 IU/ml is now used to guide clinical decision-making for infection control measures.
In conclusion, we were able to standardize the quantifcation of two molecular assays which are based on diferent detection systems by calibrating the results to the frst international standard for SARS-CoV-2 RNA, which can lead to comparable results between assays and laboratories and can be helpful in clinical decision-making.

Data Availability
All data used to support the fndings of this study are available from the corresponding author on request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.